Material type: BookPublisher: 1997Subject(s): Department of MicrobiologyDDC classification: 0594,T Dissertation note: Hydropericardium syndrome (HPS) an avian adenovirus infection has been identified in poultry flocks all over Pakistan. This project was designed to study various aspects of HPS in terms of its aetiology (virus isolation, purification, and propagation in vitro), route of transmission, clinical picture, and pathology. In addition, identity of the contagium was confirmed through immunofluorescence, electron microscopy, biological titration, serotyping and molecular characterization. Attempts were also made to develop improved vaccines against the FIPS virus.
Investigation on purified HPS agent indicated that this isolate was a new avian adenovirus (AAV) pathotype belonging to serotype-4 of group-I. This isolate was named after Pakistan Agricultural Research Council-I as PARC-I isolates. The results of one-way virus neutralization test with reference AAV antisera (1-1 1) confirmed that the isolate designated as PARC-I isolate belonged to serotype-4. The AAV serotype-4 isolate has the potential to produce the immunoprecipitating and virus neutralizing antibodies.
It was observed that this isolate is capable of causing 1-IPS in broiler chicks. The lesions caused by this virus were identical to those of HPS observed under the field outbreaks. In embryonated chicken eggs, the isolate causes mortality, generalized mu scular congestion, hepatitis and stunted embryonic growth. This virus also causes typical cytopathic effects: rounding, moderate swelling and grape-like clustering upon inoculation onto chicken embryo liver (CEL) cells. The biological characterization indicated that the isolate PARC-I possessed standard properties of other known serotypes of AAV. Although, the new isolate is biologically and serologically identical to serotype-4 of AAV, it is more virulent than the previously known strains of serotype-4. The present work has further indicated that isolates obtained from different TIPS outbreaks over the last 10 years have identical in vitro and in vivo characteristics.
The polypeptide analysis using SDS-PAGE confirmed the identity of i-IPS virus as AAV. The protein pattern of prototype strain of serotype-4 is quite comparable with those of the new isolate. The protein Profile of the isolate PARC-i and eleven other AAV serotypes were also compared. The results indicated that there were seven dense identifiable protein bands on the gel. These virus polypeptides were designated as II, Ill, lIla, IV, IVa, V and VI with molecular weights of 120 Kd, 86 Kd, 65 Kd, 55 Kd, 48 Kd, 42 Kd and 24 Kd, respectively.
Western blotting was also performed to identify common immunogenic antigen (s) amongst the PARC-I isolate and other AAV serotypes of group- I. A total of 7 common bands of the same MW as seen in the gel were detectable in the lane of PARC-i isolate and the lanes of serotypes 1-1 1. In PARC-i lanes, one band above 120 Kd was seen reacting to the hyperimmune serum whereas, 2-3 such bands were detectable in other 1-I I serotypes ol' avian adenoviruses. The western blot studies indicated that at least five of the major proteins are conserved in the eleven AAV serotypes and PARC-i isolate. Although minor antigenic variations among different avian adenovirus serotypes existed no significant differences in the immunogenic proteins, among the eleven adenovirus serotypes, were observed except serotype-9, where 24 Kd band was uniquely present. The sharing of common antigens in various serotypes especially between serotype-9 and PARC-i isolate indicated that this serotype might be useful for developing heterotypic vaccine against HPS, as many field reports indicate failure of currently used vaccines to confer effective resistance in chickens especially 3-4 wks post FIPSV vaccination. One of the reasons of the persistence of HPS might be due to the absence of maternal immunity in broilers, as the breeders are neither properly immunized nor hyperimmunized against HPS.
All the four experimental vaccines provoked almost similar level of protection in the inoculated broiler chicks as they resisted virulent HPSV challenge on 25th day postvaccination. A decrease in protection levels from days 32 postchallenge onwards was evidenced by decrease in the corresponding antibody titre in the vaccinated chicks.

Hydropericardium syndrome (HPS) an avian adenovirus infection has been identified in poultry flocks all over Pakistan. This project was designed to study various aspects of HPS in terms of its aetiology (virus isolation, purification, and propagation in vitro), route of transmission, clinical picture, and pathology. In addition, identity of the contagium was confirmed through immunofluorescence, electron microscopy, biological titration, serotyping and molecular characterization. Attempts were also made to develop improved vaccines against the FIPS virus.

Investigation on purified HPS agent indicated that this isolate was a new avian adenovirus (AAV) pathotype belonging to serotype-4 of group-I. This isolate was named after Pakistan Agricultural Research Council-I as PARC-I isolates. The results of one-way virus neutralization test with reference AAV antisera (1-1 1) confirmed that the isolate designated as PARC-I isolate belonged to serotype-4. The AAV serotype-4 isolate has the potential to produce the immunoprecipitating and virus neutralizing antibodies.

It was observed that this isolate is capable of causing 1-IPS in broiler chicks. The lesions caused by this virus were identical to those of HPS observed under the field outbreaks. In embryonated chicken eggs, the isolate causes mortality, generalized mu scular congestion, hepatitis and stunted embryonic growth. This virus also causes typical cytopathic effects: rounding, moderate swelling and grape-like clustering upon inoculation onto chicken embryo liver (CEL) cells. The biological characterization indicated that the isolate PARC-I possessed standard properties of other known serotypes of AAV. Although, the new isolate is biologically and serologically identical to serotype-4 of AAV, it is more virulent than the previously known strains of serotype-4. The present work has further indicated that isolates obtained from different TIPS outbreaks over the last 10 years have identical in vitro and in vivo characteristics.
The polypeptide analysis using SDS-PAGE confirmed the identity of i-IPS virus as AAV. The protein pattern of prototype strain of serotype-4 is quite comparable with those of the new isolate. The protein Profile of the isolate PARC-i and eleven other AAV serotypes were also compared. The results indicated that there were seven dense identifiable protein bands on the gel. These virus polypeptides were designated as II, Ill, lIla, IV, IVa, V and VI with molecular weights of 120 Kd, 86 Kd, 65 Kd, 55 Kd, 48 Kd, 42 Kd and 24 Kd, respectively.

Western blotting was also performed to identify common immunogenic antigen (s) amongst the PARC-I isolate and other AAV serotypes of group- I. A total of 7 common bands of the same MW as seen in the gel were detectable in the lane of PARC-i isolate and the lanes of serotypes 1-1 1. In PARC-i lanes, one band above 120 Kd was seen reacting to the hyperimmune serum whereas, 2-3 such bands were detectable in other 1-I I serotypes ol' avian adenoviruses. The western blot studies indicated that at least five of the major proteins are conserved in the eleven AAV serotypes and PARC-i isolate. Although minor antigenic variations among different avian adenovirus serotypes existed no significant differences in the immunogenic proteins, among the eleven adenovirus serotypes, were observed except serotype-9, where 24 Kd band was uniquely present. The sharing of common antigens in various serotypes especially between serotype-9 and PARC-i isolate indicated that this serotype might be useful for developing heterotypic vaccine against HPS, as many field reports indicate failure of currently used vaccines to confer effective resistance in chickens especially 3-4 wks post FIPSV vaccination. One of the reasons of the persistence of HPS might be due to the absence of maternal immunity in broilers, as the breeders are neither properly immunized nor hyperimmunized against HPS.

All the four experimental vaccines provoked almost similar level of protection in the inoculated broiler chicks as they resisted virulent HPSV challenge on 25th day postvaccination. A decrease in protection levels from days 32 postchallenge onwards was evidenced by decrease in the corresponding antibody titre in the vaccinated chicks.

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